Abstract Enabling access to comprehensive biomedical information is central to the mission of the National Library of Medicine and key to disease prevention for better health outcomes nationally and globally. In the area of infectious disease prevention, real time and non-biased sequence data are essential for early detection of threats to mitigate outbreaks, epidemics and pandemics. This proposed research project aims to innovate in the creation and accessibility of metagenomic sequence data relating to infectious agents by leveraging the potentially transformative tool of wastewater-based epidemiology (WBE). The WBE approach employs analysis of community wastewater from large populations to enable non-invasive, near real-time monitoring of both symptomatic and asymptomatic individuals. When combined with high-throughput metagenomic sequencing (HTS), the resulting WBE-HTS approach is promising for detecting a broad spectrum of infectious agents simultaneously without any a priori assumptions about virus identity or presence. This project will test the hypothesis that the processing of aggregated metagenomic sequence data on viruses detectable in U.S. wastewater can serve to identify vulnerabilities, outbreaks and epidemics early, thereby creating a novel and potentially life-saving diagnostic approach for protecting public health. For hypothesis testing, the team will leverage Arizona State University's Human Health Observatory (HHO), the largest sewage monitoring network and largest archive of sewage and sludge samples worldwide, representing more than 200 U.S. cities and over 300 cities globally. The overall goal of this work is to develop a broadly applicable online bioinformatics framework and to demonstrate its utility in a case study employing WBE and HTS. The three specific aims are to: (i) inventory viruses extant in the U.S. in a comprehensive database, using existing database sources and WBE; (ii) generate a knowledge repository and online portal of metagenomic data to support near real-time surveillance of the human?associated virome, and (iii) evaluate the utility of the WBE bioinformatics framework for tracking viral outbreaks and epidemics. Successful completion of this project will provide the U.S. with an early warning system for pathogen detection and outbreak tracking that can serve to prevent epidemics, thereby reducing morbidity and mortality in the U.S. from contagious pathogens, first for viruses and potentially in the future for pathogenic bacteria and other biohazards.